The coronavirus mouse hepatitis virus, strain A59 (MHV-A59), causes acute encephalitis and hepatitis followed by chronic demyelination in mice. Infection of mice by MHV provides a model system for the study of human demyelinating diseases such as multiple sclerosis. The long term goal of this project is to understand the molecular basis of the biological properties of MHV-A59. The focus of this proposal is on the activities encoded in gene 1 of MHV-A59, the putative polymerase gene. This 21 kb gene encodes three predicted proteinase domains as well as polymerase, NTPase/helicase and zinc finger domains. The gene contains two large open reading frames (ORF 1a and ORF 1b); ORF 1b is thought to be expressed via a translational frame shift at the end of ORF 1a. The presence of large ORFs and proteinase motifs suggests that synthesis and processing of the viral replicase will involve a cascade of proteolytic events. The goal of this project is to study the structure and function as well as the mechanism of genesis of the polypeptides encoded in the putative RNA polymerase gene.The focus of this proposal will be on the three predicted proteinase domains in ORF 1a (1) cDNA clones containing portions of ORF 1a will be used to analyze the two predicted papain-like proteinase domains, PLP-1 and PLP-2. N-terminal protein sequencing will be carried out to precisely map the recognition/cleavage site for p28 in vivo and to map second cleavage site observed in vitro. We will determine whether the second predicted papain-like domain does indeed have proteinase activity. We will begin to explore a possible role for intracellular factors in the MHV papain-like activities.We will express in E. coli high levels of PLP-1 and begin to characterize its enzymatic properties on peptide substrates. (2) The cDNA clone containing the predicted "picornavirus 3C-like" proteinase domain will be analyzed both by in vitro translation and in E. coli to verify its catalytic amino acids, and to determine its specificities for cleavage both of itself in cis and predicted cleavage of ORF 1b polypeptides. Using antibodies, intracellular forms of the 3C-like proteinase will be examined. A possible association of these polypeptides with membranes will also be investigated. The understanding of these activities will contribute greatly to understanding the molecular basis for replication and pathogenesis of MHV and furthermore, will aid in the design of antiviral strategies.